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Letters to the Editor |
1 INSERM U555, Faculté de Médecine and Hôpital Universitaire de la Timone, Marseille, France
2 University of Medicine of Damascus, Damascus, Syria
aAuthor for correspondence. E-mail bernard.mallet{at}medecine.univ-mrs.fr.
To the Editor:
Iodide plays a central role in thyroid physiology, and iodine compounds are essential for normal vertebrate growth and development. Useful information about the iodine nutritional status of a population can be obtained by measuring urinary iodine to estimate the prevalence of iodine deficiency disorders. Several methods for measuring urinary iodine are currently available [for a review, see Ref. (1)]: most of these involve the Sandell–Kolthoff reaction (2), which is based on a colorimetric ceric–arsenic assay. However, these methods are susceptible to several problems: e.g., various contaminants can cause the reduction of cerium(IV), or pigments or drug metabolites in the urine can increase the natural yellow color of the urine, leading to false-negative results. The digestion of urine samples, which is the first step in all of the methods based on the Sandell–Kolthoff reaction, is therefore crucial.
In this study, we tested three digestion methods on 200 urine samples from patients hospitalized at the La Timone University Hospital: the conventional chloric acid method (1); an acid ammonium persulfate method (3)(4); and a method involving a HNO3–HCl mixture. For the latter method, the digestion was performed by mixing 3 mL of 14.3 mol/L HNO3 with 2 mL of 12.1 mol/L HCl just before use. Urine samples (250 µL) were digested with 125 µL of the mixture for 60 min at 110 °C, then brought to 2 mL with phosphate-buffered saline (PBS), pH 9.0.
The microassay system is based on the catalytic Sandell–Kolthoff reaction, as described previously (3)(5). In this assay, the catalytic activity of iodide on the oxidation of arsenic(III) by cerium(IV) is measured by monitoring the reduction of the yellow ceric ions.
In the case of chloric acid digestion, we noted that 
75% of the urine samples had a slightly brown color. This color was probably attributable to the oxidation of iodide into iodine, and to confirm this hypothesis, we subjected 10 samples of a KI solution (10.8 µmol/L) to chloric acid digestion. All of the digested samples showed a brown color. Iodine analysis performed by titration showed that a mean (SD) of 42 (8)% of the initial iodide content was transformed into free iodine, which has no catalytic activity in the Sandell–Kolthoff reaction. Similar results were also obtained with perchloric acid digestion. In the case of ammonium persulfate digestion, 48% of the urine samples showed a yellow color. To assess the effects of this natural yellow color of the urine samples on the urinary iodine measurements, we placed 5 µL of digested urine plus 95 µL of PBS (pH 9.0) or 20 µL of digested urine plus 80 µL of PBS (pH 9.0) in wells of a 96-well microtiter plate. After we added 60 µL of arsenious acid and incubated the mixture at room temperature for 10 min, we monitored the absorbance at 405 nm (A405 nm) in the absence of ceric ammonium sulfate. With the 20-µL samples, the absorbance ranged from 0.024 to 0.209 [mean (SD), 0.126 (0.093)]. However, with the 20-µL samples, only 8% of the urine samples were slightly colored after the HNO3–HCl digestion, and none of them gave absorbance values >0.05 [mean (SD), 0.018 (0.011)]. To assess the resulting percentage error attributable to the natural yellow color of the digested urine, we measured the urinary iodide content of two aliquots (5 and 20 µL) of samples with A405 nm values >0.024. As can be seen from Table 1
, the urinary iodide content measured in 20-µL urine samples digested with ammonium persulfate was significantly lower than the values obtained with either 5-µL samples digested with ammonium persulfate or 5- and 20-µL urine samples digested with the HNO3–HCl mixture. The extent of the error resulting from the natural yellow color of the samples depended on both the iodide concentration in the microplate well and the intensity of the natural yellow color. Accordingly, for the 5-µL sample, the natural yellow color of the urine samples digested with ammonium persulfate gave a relatively low absorbance value [mean (SD), 0.021 (0.016)], which did not affect the loss of the yellow color of cerium(IV) attributable to the catalytic activity of the iodide. A similar conclusion can be reached about the digestion method involving the use of the HNO3–HCl mixture because the absorbance of the natural yellow color of the digested urine samples was suitably low, regardless of the volume used.
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This is the first time to our knowledge that the problem of the natural yellow color of urine samples has been investigated in connection with the determination of urinary iodine. The presence of the yellow color leads to underestimation of the urinary iodide concentration. Therefore, when testing a population with a normal iodine diet, the results obtained with the two most commonly used digestion methods will be consistent if the volume of sample in the well is not >5 µL.
In addition to urinary iodide concentration assessments, there are other fields in which iodine measurements are required: the methods used to measure the iodine present in human tissues and food products, for example, must ensure complete digestion without the production of a yellow color in the digested products. For these applications, only the HNO3–HCl digestion method, which yields a moderate yellow color, if any, is compatible with larger sample volumes of up to 40 µL (5)(6); it also shows greater sensitivity, which makes it possible to analyze trace amounts of iodine in these samples.
In conclusion, the ammonium persulfate method for urine digestion is advantageous from the point of view of its safety and ease of operation, but close attention must be paid to the volume of the urine sample used, which may limit the sensitivity of the analysis. On the other hand, although the HNO3–HCl digestion method has most of the drawbacks associated with the use of concentrated acids (e.g., it is hazardous and requires the use of an expensive fume hood), its accuracy is only slightly affected by the natural yellow color of the digested urine samples, regardless of the sample volume tested. The method based on the HNO3–HCl mixture could provide a particularly useful means of assessing the iodine content in various media, and there exist many potential applications for this efficient digestion process.
References
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